JP7000315B2 - Highly bound active HPV T cell receptor - Google Patents

Description

The present invention relates to novel highly binding active antigen recognition constructs for human papillomavirus antigens. The present invention provides a novel T cell receptor (TCR) -based molecule that is selectively and specific for the HPV 16/18 proteins E5, E6 and E7. The TCR of the present invention, and HPV antigen-binding fragments derived thereto, are not only used for diagnosis, treatment and prevention of HPV infection, but also cancer caused by HPV infection as a secondary disease mediated by HPV infection, for example. , Cervical cancer, nasopharyngeal cancer, head and neck cancer, etc. Used for diagnosis, treatment and prevention. Further provided are nucleic acids encoding the proteins of the invention and recombinant cells expressing them.

The TCR is a heterodimeric cell surface protein of the immunoglobulin superfamily associated with the invariant protein of the CD3 complex involved in the mediation of signal transduction. There are αβ and γδ types of TCRs, which are structurally similar but have completely different anatomical sites and possibly functions. The extracellular portion of the native heterodimer αβTCR consists of two polypeptides, each with a membrane proximal constant domain and a membrane distal variable domain. Each constant domain and variable domain contains an intrachain disulfide bond. Variable domains contain highly polymorphic loops that resemble the complementarity determining regions (CDRs) of antibodies. The use of TCR gene therapy overcomes many current hurdles. This makes it possible to give the patient's own T cells the desired specificity, generate a sufficient number of T cells in a short period of time, and avoid their depletion. TCRs can be converted to central memory T cells or T cells with stem cell characteristics to further ensure persistence and function during transfer. TCR-engineered T cells are injected into patients who cause lymphopenia by chemotherapy or irradiation, allowing efficient engraftment but inhibiting immunosuppression.

Human papillomavirus (HPV) is a small, non-enveloped double-stranded DNA virus that infects the skin and / or mucosal epithelium. It is known that there are over 100 HPV genotypes. A subset of mucous secretory HPV that infects the anal genital tract of men and women is the most common sexually transmitted human pathogen. These sexually transmitted mucosal affinity HPVs are classified as high risk (HPV16 and HPV18) or low risk (HPV6 and HPV11) depending on their carcinogenicity. High-risk genotypes are causally associated with anal genital cancer, including nearly 100% of cervical cancers, which is the second leading cause of cancer death in women worldwide.

Persistence of carcinogenic HPV is required for precervical cancer and the development of cancer. However, the factors that determine the persistence and progression of tumorigenicity of the virus are not fully understood. The initial event of cervical carcinogenesis after viral infection depends on the fact that high-risk HPV types undergo specific changes that abolish the transcriptional regulation of viral gene expression in infected keratinocytes. Inactivation of these cell regulatory functions allows transcriptional deregulation of the early viral genes E6 and E7, thereby causing cell proliferation, inhibition of apoptosis, differentiation reprogramming and chromosomal instability. These changes can support the integration of the episome HPV genome into the chromosomes of host cells and contribute to further overexpression of the viral genes E6 and E7, E7 cancer protein levels in the early stages of cervical carcinogenesis. Brings a rise in.

The crucial importance of viral cancer proteins E6 and E7 in carcinogenesis has been further demonstrated by the ability of high-risk E7 proteins to work with high-risk E6 to efficiently immortalize primary human keratinocytes in vitro. .. In addition, consistent overexpression of the E6 and E7 carcinogenic genes is required to induce and maintain the transformed phenotype of cervical cancer cells.

Therefore, detection of E6 and E7 proteins appears to be an excellent diagnostic tool. Various antibodies are already known in the art, but they have been described as exhibiting low sensitivity or low specificity or cross-reactive with various HPV-type E7 proteins. Also, there are differences between batches because polyclonal antibodies can only be produced in limited quantities in a single animal. The difficulty in producing highly specific and sensitive monoclonal antibodies to the E7 protein is primarily due to the low immunogenicity of the E7 protein.

Therefore, one of the objects of the present invention is to overcome the above-mentioned difficulties and is very specific and higher for cost-effective, rapid and reliable diagnosis of HPV infection. It is to provide a sensitive means, eg, a means through the detection of the presence or absence of an early HPV protein such as E5, E6 or E7.

Effective prophylactic vaccines against the two most common high-risk HPVs are available, but they are expensive, social acceptance problems, and HPV-related anal genital cancer are the most common, especially developing. There are limits to the vaccination available to women in the country that may limit the availability of this vaccine. Therefore, there remains a need to clarify direct treatments for targeting cancers resulting from HPV infections and HPV infections such as cervical cancer. Therefore, another technical challenge to be solved by the present invention is to provide an efficient and highly binding active T cell receptor-based molecule used in therapeutic approaches in the treatment and prevention of HPV-mediated diseases. Is

The above task is an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity with the amino acid sequence selected from SEQ ID NOs: 9 to 28. It is solved in the first aspect by an antigen recognition construct comprising.

SEQ ID NOs: 9-28 correspond to the TCR T1-T9 CDR3 regions shown in the tables in the Examples (Examples 1-9) sections of the present application. The HPV-responsive TCR of the present invention was a surprising discovery as it was highly avid compared to the state-of-the-art TCR for HPV antigens. In some cases, the invention is more specific, more sensitive, more selective, and / or more avid compared to the state-of-the-art TCR. In a preferred embodiment of the invention, the antigen recognition constructs are amino acid sequences selected from SEQ ID NOs: 9-28 and at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100. Includes complementarity determining regions 3 (CDR3) with% sequence identity.

CDR3 of the antigen recognition construct of the present invention may be mutated. Mutations in the CDR3 sequences of SEQ ID NOs: 9-28 preferably include substitutions, deletions, additions, or insertions of 3 or less, preferably 2 or less, most preferably one amino acid residue.

In another further or alternative embodiment, the antigen recognition construct is an HPV antigen recognition construct.

In another further or alternative embodiment, the antigen recognition construct may further comprise the CDR1 and CDR2 domain sequences. Within the variable domain, CDR1 and CDR2 are found in the variable (V) region of the polypeptide chain, and CDR3 covers part of the variable (V) region and all of the diversity (D) and joining (J) regions. include. CDR3 is the most variable and is the major CDR involved in antigen recognition. The CDR1 and CDR2 sequences can be selected from the CDR sequences of the human variable chain allele.

The native alpha-beta heterodimer TCR has alpha and beta chains. Each chain contains a variable region, a joining region and a stationary region, and the beta chain usually contains a short diversity region between the variable region and the joining region, but this diversity region is often regarded as part of the joining region. Is done. Each variable region contains three CDRs (complementarity determining regions) embedded in the framework sequence, one of which is a high frequency variable region called CDR3. There are several alpha chain variable (Vα) regions and several beta chain variable (Vβ) regions, distinguished by framework regions, CDR1 and CDR2 sequences, and partially defined by CDR3 sequences. The Vα type is referred to by a unique TRAV number in IMGT nomenclature, and the Vβ type is referred to by a unique TRBV number.

Thus, in another or alternative embodiment, the antigen recognition construct of the invention comprises the combinations CDR1, CDR2, CDR3 provided in the table of Examples section where the respective variable chain alleles are shown along with the CDR3 sequence. Therefore, preferred antigen recognition constructs of the invention include at least one, preferably all CDR sequences, CDR1, CDR2 and CDR3.

In one embodiment of the invention, the antigen recognition construct specifically binds to the human papillomavirus (HPV) antigen, preferably the HPV antigen is a protein selected from HPV16 E5, HPV16 E6, HPV16 E7, HPV18 E6 and HPV18 E7. be.

As used herein, the term "specificity" or "antigen property" or "specific" given to an antigen means that the antigen recognition construct is specific to said antigen, preferably to HPV antigen, more preferably to high binding activity. It means that it can be combined with. For example, low concentrations of T cells expressing TCR (eg, about 10 ^-11 mol / l, 10 ^-10 mol / l, 10 ^-9 mol / l, 10 ^-8 mol / l, 10 ^-7 mol / l, Target cells pulsed with 10 ^-6 mol / l, 10 ^-5 mol / l HPV antigens (eg, HPV16 E5, E6 and E7 antigens) At least about 200 pg / ml or more when co-cultured with target cells (eg, HPV16 E5, E6 and E7 antigens) For example, 250 pg / ml or more, 300 pg / ml or more, 400 pg / ml or more, 500 pg / ml or more, 600 pg / ml or more, 700 pg / ml or more, 1000 pg ml or more, 2,000 pg / ml or more, 2,500 When secreting interferon γ (IFN-γ) of pg / ml or more and 5,000 pg / ml or more), TCR can be considered to have “antigen specificity” for HPV antigen. Alternatively or additionally, TCR-expressing T cells are at least twice as high as the non-transfection background level of IFN-γ when co-cultured with target cells pulsed with low concentrations of HPV antigen. When secreting -γ, the TCR can be considered to have "antigen specificity" for HPV. Such "specificity" described herein can be analyzed, for example, by ELISA.

In an alternative or alternative embodiment of the invention, the antigen recognition construct selectively binds to the human papillomavirus (HPV) antigen, preferably the HPV antigen is selected from HPV16 E5, HPV16 E6, HPV16 E7, HPV18 E6 and HPV18 E7. It is a protein.

The terms "selectivity" or "selective recognition / binding" preferably selectively recognize or bind only one specific epitope and preferably show no or substantially cross-reactivity to another epitope. Not shown, understood to mean the properties of an antibody or T cell receptor. Preferably, "selectivity" or "selective recognition / binding" is such that the antigen recognition construct (eg, TCR) selectively recognizes or binds to only one specific epitope, preferably to another epitope. Means that they do not show or substantially do not show cross-reactivity, such that the epitope does not show or substantially does not show cross-reactivity to another epitope and another protein by the antigen recognition construct. In addition, it is unique to one protein.

The antigen recognition construct according to the present invention is preferably selected from an antibody or a derivative thereof or a fragment thereof, or a T cell receptor (TCR) or a derivative thereof or a fragment thereof. The antibody or derivative or fragment of the TCR of the present invention preferably retains the binding / recognition ability of the original molecule to the antigen, particularly the specificity and / or selectivity as described above.

In one aspect of the invention, the TCR of the invention is capable of recognizing HPV antigens in a manner that depends on major histocompatibility complex (MHC) class I. As used herein, the term "MHC class I dependent embodiment" means that TCR induces an immune response when it binds to the HPV antigen under certain circumstances of MHC class I molecules. The MHC class I molecule may be any MHC class I molecule known to those of skill in the art, including, for example, the HLA-A molecule. In a preferred embodiment of the invention, the MHC class I molecule is an HLA-A molecule, preferably HLA-A2.01.

The following epitopes (SEQ ID NOs: 1-9) were used to produce the TCR of the Examples: YIIFVYIPL (HPV 16 E5 _63-71 ), KLPQLCTEL (HPV 16 E6 _11-19 ), TIHDIILECV (HPV 16 E6 ₂₉ ). _-38 ), YMLDLQPET (HPV 16 E7 _11-19 ), YMLDLQPETT (HPV 16 E7 _11-20 ), TLGIVCPI (HPV 16 E7 _86-93 ), KCIDFYSRI (HPV 18 E6 _67-75 ), FQQLFLNTL (HPV 18 E7 ₈₆ ) _-94 ). Thus, in one aspect of the invention in which the antigen recognition construct is described, the antigen recognition construct is specific and / or selective for one of the HPV epitopes. It is to those of skill in the art that the antigen recognition constructs of the invention specifically and / or selectively bind to their respective corresponding epitopes, depending on which CDR3 is present, as described in the Examples section of the present application. it is obvious.

The present invention provides both single-stranded antigen recognition constructs and double-stranded antigen recognition constructs.

The present invention specifically provides a TCR as an antigen recognition construct or a fragment thereof or a derivative thereof. The TCR is preferably human, and it is understood that the TCR is produced from the human TCR locus, and from this it is understood to contain the human TCR sequence. Furthermore, the TCRs of the present invention are of human origin and can be characterized by specifically recognizing HPV antigens. The term "origin" in this regard refers to a mutated sequence derived from each "original" sequence but containing amino acid modifications. For example, a TCR of human origin may contain a TCR sequence of a non-human species or may be artificially introduced into a human TCR sequence.

Another additional or alternative embodiment of the invention provides the antigen recognition construct that induces an immune response, preferably characterized by increasing interferon (IFN) γ levels.

The TCR of the present invention may be provided as a single-stranded α molecule or a single chain β molecule, or as a double chain construct composed of both α chain and β chain, or both γ chain and δ chain. Therefore, the TCR of the present invention is one amino acid sequence selected from SEQ ID NOs: 9, 10, 12, 14, 16, 18, 21, 23, 25, 27 and at least 50%, 60%, 70%, 80%, Alpha chain CDR3 with 90%, 95%, 98%, 99%, or 100% sequence identity and / or SEQ ID NOs: 11, 13, 15, 17, 19, 20, 22, 24, 26, 28. It preferably comprises beta chain CDR3 having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity with the amino acid sequence selected from.

Preferably, the double chain TCR or an antigen-binding fragment thereof comprises the amino acid sequence set forth in SEQ ID NO: 9 in the first polypeptide chain and the amino acid sequence set forth in SEQ ID NO: 11 in the second polypeptide chain. ; Or, in the first polypeptide chain, the amino acid sequence shown in SEQ ID NO: 10 is contained, and in the second polypeptide chain, the amino acid sequence shown in SEQ ID NO: 11 is contained; or in the first polypeptide chain. Includes the amino acid sequence set forth in SEQ ID NO: 12 and comprises the amino acid sequence set forth in SEQ ID NO: 13 in the second polypeptide chain; or comprises the amino acid sequence set forth in SEQ ID NO: 14 in the first polypeptide chain. And, in the second polypeptide chain, the amino acid sequence set forth in SEQ ID NO: 15; or in the first polypeptide chain, the amino acid sequence set forth in SEQ ID NO: 16 and in the second polypeptide chain. Includes the amino acid sequence set forth in SEQ ID NO: 17; or contains the amino acid sequence set forth in SEQ ID NO: 18 in the first polypeptide chain and contains the amino acid sequence set forth in SEQ ID NO: 19 in the second polypeptide chain. ; Or, in the first polypeptide chain, the amino acid sequence shown in SEQ ID NO: 18 is contained, and in the second polypeptide chain, the amino acid sequence shown in SEQ ID NO: 20 is contained; or in the first polypeptide chain. Includes the amino acid sequence set forth in SEQ ID NO: 21 and comprises the amino acid sequence set forth in SEQ ID NO: 22 in the second polypeptide chain; or comprises the amino acid sequence set forth in SEQ ID NO: 23 in the first polypeptide chain. And, in the second polypeptide chain, the amino acid sequence set forth in SEQ ID NO: 24; or in the first polypeptide chain, the amino acid sequence set forth in SEQ ID NO: 25, and in the second polypeptide chain. Containing the amino acid sequence set forth in SEQ ID NO: 26; or comprising the amino acid sequence set forth in SEQ ID NO: 27 in the first polypeptide chain and comprising the amino acid sequence set forth in SEQ ID NO: 28 in the second polypeptide chain. .. Any one of the double chain TCRs or antigen-binding fragments thereof is the preferred TCR of the present invention. In some embodiments, the CDR3 of the double chain TCR of the invention may be mutated. The mutations in CDR3 of SEQ ID NOs: 9-28 provided above include substitutions, deletions, additions, or insertions of three or less, preferably two, most preferably one amino acid residues. In some embodiments, the first polypeptide chain may be a TCRα or γ chain and the second polypeptide chain may be a TCRβ or δ chain. The TCR is preferably a combination of αβ or γδ.

The TCR or its antigen-binding fragment is composed of a TCRα chain and a TCRβ chain, or a γ chain and a δ chain. Such a double-stranded TCR contains a variable region containing the CDR1 sequence, the CDR2 sequence and the CDR3 sequence within each strand. The TCRs are SEQ ID NO: 29 and SEQ ID NO: 31 (T1a); or SEQ ID NO: 30 and SEQ ID NO: 31 (T1b); or SEQ ID NO: 32 and SEQ ID NO: 33 (T2); or SEQ ID NO: 34 and SEQ ID NO: 35 (T3); or SEQ ID NO: 36 and SEQ ID NO: 37 (T4); or SEQ ID NO: 38 and SEQ ID NO: 39 (T5a); or SEQ ID NO: 38 and SEQ ID NO: 40 (T5b); or SEQ ID NO: 41 and SEQ ID NO: 42 (T6); or SEQ ID NO: 43 and SEQ ID NO: 44 (T7); or SEQ ID NO: 45 and SEQ ID NO: 46 (T8); or CDR1-3 contained in the variable chain amino acid sequences set forth in SEQ ID NO: 47 and SEQ ID NO: 48 (T9).

Some aspects of the invention relate to a TCR or fragment thereof composed of a TCRα chain and a TCRβ chain, wherein the TCR is the α chain and the β chain, SEQ ID NO: 29 and SEQ ID NO: 31 (T1a); or SEQ ID NO: respectively. 30 and SEQ ID NO: 31 (T1b); or SEQ ID NO: 32 and SEQ ID NO: 33 (T2); or SEQ ID NO: 34 and SEQ ID NO: 35 (T3), respectively; or SEQ ID NO: 36 and SEQ ID NO: 37 (T4); or respectively. SEQ ID NO: 38 and SEQ ID NO: 39 (T5a); or SEQ ID NO: 38 and SEQ ID NO: 40 (T5b), respectively; or SEQ ID NO: 41 and SEQ ID NO: 42 (T6); or SEQ ID NO: 43 and SEQ ID NO: 44 (T7), respectively; Alternatively, it comprises the variable region sequences of the α and β chains set forth in SEQ ID NO: 45 and SEQ ID NO: 46 (T8); or SEQ ID NO: 47 and SEQ ID NO: 48 (T9), respectively.

The TCRs of the invention may further comprise constant regions derived from any suitable species, such as humans or mice. In one aspect of the invention, the TCR of the invention further comprises a human constant region. In some preferred embodiments, the constant region of the TCR of the invention may be modified slightly, for example by introducing a mouse sequence that may increase the stability of the TCR.

As used herein, an antigen recognition construct, or TCR, or any of the TCRs described herein (eg, such as complementarity determining regions (CDRs), variable regions, constant regions, α chains, and / or β chains). When referring to a component of, the term "mouse" or "human" means a TCR (or component thereof) derived from an unreconstructed TCR locus of mouse or human, respectively.

In one aspect of the invention, the chimeric TCR can include the α chain of the TCR and the β chain of the TCR. Each of the α-chain and β-chain of the chimeric TCR of the present invention can independently contain an arbitrary amino acid sequence. Preferably, as described above, the α chain comprises a variable region of the human α chain and a constant region of the mouse α chain.

One aspect of the TCR of the present invention includes a human variable region and a human constant region according to the above aspect.

The TCR of the present invention may be provided as a single-stranded TCR (scTCR). The scTCR may include a polypeptide in the variable region of the first TCR chain (eg, α chain) and a polypeptide of the entire (overall) second TCR chain (eg, β chain), or vice versa. In addition, the scTCR can optionally include one or more linkers that link the two or more polypeptides together. As described herein, the linker can be, for example, a peptide that links two single strands. As an example of scTCR of the present invention, it is also provided as a fusion with a human cytokine such as IL-2, IL-7 or IL-15.

The antigen recognition construct according to the present invention can also be provided in the form of a multimeric complex containing at least two scTCR molecules, each of which is fused to at least one biotin moiety. The scTCR molecules are interconnected by a biotin-streptavidin interaction, allowing the formation of the multimer complex. Similar approaches to the generation of multimer TCRs are possible and are included in the present disclosure. Also provided are higher order multimer complexes containing more than two scTCRs of the invention.

For the purposes of the present invention, a TCR is a portion having at least one TCR alpha and / or TCR beta variable domain. In general, these include both TCR alpha variable domains and TCR β variable domains. These may be in the αβ heterodimer or single-stranded form. For use in adoptive immunotherapy, the αβ heterodimer TCR may be transfected, for example, with a full-length chain having both a cytoplasm and a transmembrane domain. If desired, there may be disulfide bonds introduced between the residues of each constant domain.

In one additional preferred embodiment of the first aspect of the invention, the antigen recognition construct is the TCR. The TCR preferably comprises at least one alpha and / or beta TCR chain, wherein said TCR is the amino acid sequence set forth in any one of the TCR chains set forth in SEQ ID NOs: 22-39, or set forth in SEQ ID NOs: 22-39. Contains an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% identity with the amino acid sequence.

In a preferred embodiment, the antigen recognition construct is a human TCR or fragment thereof or a derivative thereof. A human TCR or fragment thereof or a derivative thereof is a TCR containing more than 50% of the corresponding human TCR sequence. Preferably, only a small portion of the TCR sequence is of artificial origin or of other species. However, for example, human-derived chimeric TCRs with mouse sequences in the constant domain are known to be advantageous. Therefore, particularly preferred is the TCR according to the invention, which comprises a mouse sequence in the extracellular portion of those constant domains.

As described above, it is also preferable that the antigen recognition construct of the present invention can recognize the antigen in a human leukocyte antigen (HLA) -dependent manner, preferably in an HLA-A02-dependent manner. The term "HLA-dependent mode" in the context of the present invention means that an antigen recognition construct binds to an antigen only if the antigen peptide is presented by HLA.

The antigen recognition construct in one embodiment of the invention is preferably characterized by inducing an immune response, preferably by increasing interferon (IFN) γ levels.

What is provided herein is, for example, the functional portion of any one of the TCRs (or functional variants thereof) of the present disclosure, such as any one of T1 to T9 of the TCR provided in the Section of Examples of the present application. It is also a polypeptide containing. As used herein, the term "polypeptide" refers to a single chain of amino acids comprising an oligopeptide and linked by one or more peptide bonds. With respect to the polypeptides of the invention, the functional portion of the TCR (or functional variant thereof) that is the functional portion if it specifically binds to the HPV antigens set forth in SEQ ID NOs: 1-8. It can be any moiety that contains continuous amino acids. The term "functional portion" as used with respect to TCR (or a functional variant thereof) refers to any portion or fragment of the TCR (or functional variant thereof) of the invention, which portion or fragment. It retains the biological activity of a portion of it (its parent TCR or its parent functional variant), TCR (or its functional variant). The functional part retains, for example, the ability to specifically bind to HPV antigens (eg, SEQ ID NOs: 1-8) (eg, in an HLA-A2-dependent manner), or the ability to detect, treat, or prevent cancer. The portion of the TCR (or functional variant thereof) that retains the above is included to a degree similar to, similar to, or higher than that of the parent TCR (or functional variant thereof). With respect to the parent TCR (or its functional variant), its functional portion is, for example, about 10%, 25%, 30%, 50%, 68%, 80 of the parent TCR variable sequence (or its functional variant). Can include%, 90%, 95% or more.

The functional moiety may contain additional amino acids not found in the amino acid sequence of the parent TCR or its functional variant at the amino or carboxy terminus of that moiety, or both. Desirably, the additional amino acid does not interfere with the biological function of the functional part, eg, specifically binding to the HPV antigen, and / or has the ability to detect, treat or prevent cancer or the like. More preferably, the additional amino acid enhances the biological activity as compared to the biological activity of the parent TCR or its functional variant.

Polypeptides are functional portions of either or both of the α and / or β chains of the TCR of the invention or its functional variants, eg, the α and / or β chains of the TCR of the invention or its functional variants. Can include a functional portion, including one or more CDR1, CDR2 and CDR3 of the variable region of. In one aspect of the invention, the polypeptide can comprise a functional portion comprising the amino acid sequence of SEQ ID NOs: 9-28 (CDR3 of the variable region of the TCR of the invention) or a combination thereof. In one aspect of the invention, the polypeptide of the invention can include, for example, variable regions of the TCRs of the invention or functional variants thereof, including the combinations of CDR regions described above. In this regard, the polypeptide can comprise any of the amino acid sequences of SEQ ID NOs: 29-48 (variable regions of the α or β chain).

Optionally, the antigen recognition construct of the present invention may comprise one or two polypeptides comprising any one of the sequences set forth in SEQ ID NOs: 9-48 (CDR sequence or fully variable region), or functional fragments thereof. Well, it may further comprise another amino acid sequence, eg, an amino acid sequence encoding an immunoglobulin or portion thereof, in which case the protein of the invention can be a fusion protein. In this regard, the invention provides a fusion protein comprising at least one polypeptide of the invention and at least one other polypeptide in the context of the present specification. The other polypeptide may exist as a separate polypeptide of the fusion protein, or as a polypeptide expressed in frame (tandem) with one of the polypeptides of the invention described herein. .. The other polypeptide is any peptide or protein including, but not limited to, immunoglobulins, CD3, CD4, CD8, MHC molecules, CD1 molecules (eg, CD 1a, CD 1b, CD 1c, CD 1d, etc.). It can encode a molecule or part thereof.

The fusion protein can include one or more copies of the polypeptide of the invention and / or one or more copies of the other polypeptide. For example, the fusion protein may include 1, 2, 3, 4, 5 or more copies of the polypeptide of the invention and / or other polypeptides. Suitable methods for making fusion proteins are known in the art and include, for example, recombinant methods. In one aspect of the invention, the TCR (and its functional portions and functional variants thereof), polypeptides, and proteins of the invention are single comprising a linker peptide linking an α / γ chain and a β / δ chain. Can be expressed as a protein of. In this regard, the TCRs (and their functional parts and functional variants thereof), polypeptides, and proteins of the invention contain amino acid sequences (T1-T9) of the variable regions of the TCR of the invention, and further include linker peptides. It may be included. Linker peptides can advantageously promote the expression of recombinant TCRs (and their functional parts and functional variants thereof), polypeptides, and / or proteins in host cells. The linker peptide may contain any suitable amino acid sequence. Linker sequences for single-stranded TCR constructs are well known in the art. Such single-stranded constructs may further comprise one or two constant domain sequences. Upon expression of the construct containing the linker peptide by the host cell, the linker peptide may be cleaved to give rise to the separated α and β chains.

As mentioned above, the binding functionality of the TCRs of the invention may be provided within the framework of antibodies. In its various grammatical forms, the term "antibody" is used herein to refer to an immunoglobulin molecule and an immunologically active portion of the immunoglobulin molecule, i.e., a molecule containing an antibody binding site or paratope. Will be done. Such molecules are also referred to as "antigen-binding fragments" of immunoglobulin molecules. The present invention further provides an antibody that specifically binds to the antigen described herein, or an antigen binding portion thereof. The antibody can be any type of immunoglobulin known in the art. For example, the antibody can be any isotype, such as IgA, IgD, IgE, IgG, IgM and the like. The antibody can be monoclonal or polyclonal. The antibody can be a natural antibody such as an antibody isolated and / or purified from mammals such as mice, rabbits, goats, horses, chickens, hamsters, humans and the like. Alternatively, the antibody can be a genetically engineered antibody, such as a humanized antibody or a chimeric antibody. The antibody can be in monomeric or polymer form.

The invention also provides an antigen binding portion of any of the antibodies described herein. The antigen binding moiety can be any moiety having at least one antigen binding site, such as Fab, F (ab') 2, dsFv, sFv, diabodies, and triabodies. Single-stranded variable region fragment (sFv) antibody fragments consisting of shortened Fab fragments containing the variable (V) domain of the antibody heavy chain linked to the variable (V) domain of the light chain antibody via a synthetic peptide are usually It can be prepared using the recombinant DNA technology technique of. Similarly, disulfide-stabilized variable region fragments (dsFv) can be made by recombinant DNA technology. However, the antibody fragments of the invention are not limited to these exemplary types of antibody fragments. Antibodies or antigen-binding moieties thereof are, for example, radioisotopes, fluorophores (eg, fluorescein isothiocyanate (FITC), phycoerythrin (PE)), enzymes (eg, alkaline phosphatase, horseradish peroxidase), and elementary particles (eg, alkaline phosphatase, horseradish peroxidase). For example, it can be modified to include a detectable label such as gold particles). In some cases, the CDR3 sequence of the TCR may be slightly modified, preferably by 3 or less amino acid residues, preferably by only two, and as compared to the CDR3 sequences set forth in SEQ ID NOs: 9-28. Most preferably, it may be modified at only one amino acid site. Preferably, the antibody comprises all of the CDR3, preferably CDR1 to CDR3 regions in combination, as shown for TCR T1 to T9 in the Examples section of the present disclosure.

Suitable methods for making antibodies are known in the art. For example, the standard hybridoma method is, for example, Kohler and Milstein, Eur. J. Immunol, 5, 51 1-519 (1976), Harlow and Lane (eds.), Antibodies: A Laboratory Manual, CSH Press (1988). , And CA Janeway et al. (eds.), Immunobiology, 8 Ed., Garland Publishing, New York, NY (201 1)). Alternatively, other methods include, for example, the EBV-hybridoma method (Haskard and Archer, J. Immunol. Methods, 74 (2), 361-67 (1984), and Roder et al, Methods Enzymol, 121, 140-67 ( 1986)), and a bacteriophage vector expression system (see, eg, Huse et al., Science, 246, 1275-81 (1989)) are known in the art. In addition, methods of producing antibodies in non-human animals are described, for example, in US Pat. Nos. 5,545,806, 5,569,825, and 5,714,352, and US Patent Application Publication No. 2002/0197266Al.

Some aspects of the invention also relate to soluble TCRs, TCRs or functional fragments thereof and polypeptides. As used herein, the term "soluble T cell receptor" includes the extracellular portion of the TCR α and β chains linked by disulfide bonds, but lacks the transmembrane and cytosolic domains of natural proteins, the native TCR. It is a heterodimer of the cleavage variant of. The term "soluble T cell receptor α chain sequence and soluble T cell receptor β chain sequence" refers to a TCR α chain and β chain sequence lacking a transmembrane and cytosol domain. The (amino acid or nucleic acid) sequence of the soluble TCR α-chain and β-chain may be identical to the corresponding sequence in the native TCR and is a variant of the soluble TCR α-chain and β-chain sequence compared to the corresponding native TCR. May include. The term "soluble T cell receptor" as used herein includes soluble TCRs having mutant or non-mutant soluble TCR α and β chain sequences. Variants may be present in the variable or constant regions of the soluble TCR α and β chain sequences, without limitation, without amino acid deletions, insertions, substitution mutations, and without altering the amino acid sequence. It can include changes in the nucleic acid sequence. In any case, the soluble TCRs of the present invention retain the binding function of their parent molecules.

The above-mentioned problems are also solved by the antigen recognition construct of the present invention, or the nucleic acid encoding the above-mentioned protein or polypeptide construct. The nucleic acid preferably has (a) a strand encoding the antigen recognition construct according to the invention, (b) a strand complementary to the strand of (a), or (c) (a) or ( It has a chain that hybridizes with the molecule of b) under stringent conditions. Stringent conditions are known to those of skill in the art, with particular reference to Sambrook et al, “Molecular Cloning”. The nucleic acid also optionally has additional sequences, particularly those required to express the nucleic acid sequence corresponding to the protein for expression in mammalian / human cells. The nucleic acid used may be included in a suitable vector to allow expression of the nucleic acid sequence corresponding to the peptide in the cell. However, nucleic acids can also be used to transform presenting cells (not limited to classical antigen-presenting cells such as dendritic cells) such that the nucleic acids themselves produce the corresponding proteins on the cell surface. can.

As used herein, "nucleic acid" includes "polynucleotides", "oligonucleotides" and "nucleic acid molecules" and generally means polymers of DNA or RNA. The nucleic acid is single-stranded or double-stranded and can be synthesized or obtained from a natural source (eg, isolated and / or purified). The nucleic acid can include natural, unnatural or altered nucleotides, and instead of the phosphodiesters found between the nucleotides of unmodified oligonucleotides, natural, unnatural or altered internucleotide linkages such as phosphoramide. It can include acid bonds and phosphorothioate bonds.

Preferably, the nucleic acid of the invention is recombinant. As used herein, the term "recombination" refers to (i) outside a living cell by binding a natural or synthetic nucleic acid segment to a nucleic acid molecule capable of replicating in the living cell. The constructed molecule, or (ii) a molecule resulting from the replication of the one described in (i) above. For the purposes herein, the replication can be in vitro replication or in vivo replication. Nucleic acid can include any nucleic acid sequence encoding any TCR, polypeptide, protein or functional portion or functional variant thereof described herein.

The present invention also provides a vector containing the nucleic acid of the present invention as described above. Desirably, the vector is an expression vector or a recombinant expression vector. In the context of the present invention, the term "recombinant expression vector" refers to a nucleic acid construct that allows expression of an mRNA, protein or polypeptide in a suitable host cell. The recombinant expression vector of the present invention can be any suitable recombinant expression vector and can be used to transform or transfect any suitable host. Suitable vectors include, for example, vectors designed for growth and expansion of plasmids and viruses, and / or for expression. Examples of animal expression vectors include pMP71, pEUK-Cl, pMAM, pMAMneo and pSB100Xo. Preferably, the recombinant expression vector is either a viral vector, such as a retroviral vector, or a non-viral vector, such as a transposon-based Sleeping Beauty vector. Recombinant expression vectors are specific for the type of host cell into which the vector is introduced (eg, bacteria, fungi, plants or animals) and control sequences in which the nucleic acids of the invention are expressed, such as transcription and translation initiation and. Includes stop codon. The vectors of the invention may also contain one or more marker genes that allow selection of transformed or transfected hosts. The recombinant expression vector is a natural or normative sequence operably linked to a nucleotide sequence that encodes the construct of the invention, or a nucleotide sequence that is complementary to or hybridizes to the nucleotide sequence encoding the construct of the invention. It can include promoters. Promoter selection includes, for example, strong, weak, inducible, tissue-specific and development-specific promoters. The promoter can be a non-viral promoter or a viral promoter. The recombinant expression vectors of the invention can be designed for transient expression, stable expression, or both. Recombinant expression vectors can also be made for constitutive or induced expression.

The present invention also relates to a host cell containing the antigen recognition construct according to the present invention. Specifically, the host cell of the present invention contains the above nucleic acid or vector. Host cells may be eukaryotic cells, such as plants, animals, fungi, or algae, or prokaryotic cells, such as bacteria or protozoa. The host cell may be a cultured cell or a primary cell, i.e., it may be isolated directly from an organism, such as a human. The host cell can be an attached cell or a suspended cell, i.e., a cell that proliferates in a suspension. For producing recombinant TCRs, polypeptides, or proteins, host cells are preferably mammalian cells. Most preferably, the host cell is a human cell. The host cell may be of any cell type, of any type of tissue, and at any stage of development, but is preferably peripheral blood leukocytes (PBL) or peripheral blood mononuclear cells (preferably). PBMC). More preferably, the host cell is a T cell. The T cell may be any T cell, such as a cultured T cell, such as a primary T cell, or a T cell derived from a cultured T cell lineage, such as Jurkat, SupTl, or a mammal. Preferably, it may be T cells derived from a human patient or T cells obtained from T cell progenitor cells. When obtained from mammals, T cells can be obtained from a number of sources including, but not limited to, blood, bone marrow, lymph nodes, thymus, or other tissues or body fluids. T cells may be concentrated or purified. Preferably, the T cells are human T cells. More preferably, the T cell is a T cell isolated from human. The T cells may be of any type of T cells and may be of any developmental stage, including but not limited to CD4 positive and / or CD8 positive, CD4 positive. Includes helper T cells such as Th1 and Th2 cells, CD8 positive T cells (eg, cytotoxic T cells), tumor infiltrating cells (TILs), memory T cells, naive T cells and the like. Preferably, the T cell is a CD8 positive T cell or a CD4 positive T cell.

Preferably, the host cell of the invention is a lymphocyte, preferably a T lymphocyte, eg, a CD4 or CD8 positive T cell. The host cell is preferably a tumor-reactive T cell specific for MAGE-A1-expressing tumor cells.

One further aspect of the invention relates to the antigen recognition constructs, nucleic acids, vectors, and / or host cells disclosed herein for use in pharmaceutical applications. Aspects for use in one preferred pharmaceutical use include the diagnosis, prevention and / or treatment of infectious diseases, preferably HPV infections, or proliferative diseases such as malignant or benign tumor diseases. Tumor diseases are, for example, HPV-related cancers.

The constructs, proteins, TCR antibodies, polypeptides and nucleic acids of the invention are specifically intended for use in immunotherapy, preferably adoptive T cell therapy. Administration of the compounds of the invention can include, for example, infusion of the T cells of the invention into the patient. Preferably, such T cells are patient autologous T cells transduced in vitro with the nucleic acid or antigen recognition constructs of the invention.

An object of the present invention is a method for producing a cell line expressing an HPV-specific antigen recognition construct (ARC).
An in vitro method for the production of cell lines expressing HPV-specific antigen recognition constructs (ARC).
a. The process of providing suitable host cells,
b. A step of providing a gene construct encoding ARC, wherein the ARC is at least 50%, 60%, 70%, 80%, 90%, 95 with any one amino acid sequence selected from SEQ ID NOs: 9-28. %, 98%, 99%, or 100% contains CDR3 with an amino acid sequence having sequence identity.
c. The step of introducing the gene construct into the appropriate host cell,
d. The step of expressing the gene construct in the appropriate host cell,
It can also be solved by methods, including.

In one preferred embodiment, the method may further comprise a step comprising the cell surface presentation of the ARC.

Of course, in the context of the aspect of the present invention, it is also preferable that the ARC is an ARC according to the above-mentioned aspect of the present invention. In this respect, it is further or alternatively preferred that the ARC is of mammalian, preferably human origin.

Preferred host cells for use in the methods of the invention are mammals, especially human cells such as human T cells. T cells for use in the present invention are described in detail as described above herein.

The ARC produced according to the method of the invention is a TCR in one embodiment. For example, a TCR with an additional (functional) domain or an alternative domain, eg, a TCR with a transmembrane domain as a membrane anchor, is also included. The TCR produced according to the present invention is, for example, alpha / beta TCR, gamma / delta TCR or single chain TCR (scTCR). The TCR form included in the present invention is generally any TCR known in the art, specifically the ones described above.

Desirably, the transfection system for use in the method according to the invention is a retroviral vector system. Such systems are well known to those of skill in the art.

Also included in the invention is, in one embodiment, an additional method step of purifying ARC from cells and optionally the reconstruction of translated ARC fragments in T cells.

In another aspect of the invention, T cells can be obtained or obtained by a method of producing a T cell receptor (TCR), which is specific for tumor cells and has high binding activity as described above. Such T cells depend on the host cells used in the methods of the invention, such as human or non-human T cells, preferably human TCR.

The TCRs, polypeptides, proteins (including their functional variants), nucleic acids, recombinant expression vectors, host cells (including their populations) and antibodies (including their antigen binding moieties) of the invention are isolated and / Or it can be purified. As used herein, the term "isolation" means removed from the natural environment. As used herein, the term "purification" means increased purity, where "purity" is a relative term and should not necessarily be construed as absolute purity. For example, the purity can be at least about 50%, 60%, 70%, 80%, 90%, 95% or higher, or 100%.

Antigen recognition constructs of the invention, TCRs, polypeptides, proteins (including their functional variants), nucleic acids, recombinant expression vectors, host cells (including their populations) and antibodies (including their antigen binding moieties) (including their antigen binding moieties). Hereinafter, the "TCR material of the present invention") can be formulated into a composition such as a pharmaceutical composition. In this regard, the invention relates to TCRs, polypeptides, proteins, functional moieties, functional variants, nucleic acids, expression vectors, host cells (including populations thereof), and antibodies (including antigen binding moieties thereof) and pharmaceuticals. Provided is a pharmaceutical composition comprising any of the acceptable carriers. A pharmaceutical composition of the invention containing any of the TCR materials of the invention may be a plurality of TCR materials of the invention, such as polypeptides and nucleic acids, or two or more different TCRs (functional portions thereof and functional portions thereof). (Including variants) can be included. Alternatively, the pharmaceutical composition may be another chemotherapeutic agent such as, for example, asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine and the like. Drugs or TCR materials of the invention in combination with drugs can be included, preferably the carrier is a pharmaceutically acceptable carrier. For pharmaceutical compositions, the carrier can be any of those conventionally used for the particular TCR material of the invention considered. Such pharmaceutically acceptable carriers are well known to those of skill in the art and are readily available to the public. The pharmaceutically acceptable carrier preferably has no adverse side effects or toxicity under conditions of use.

Accordingly, pharmaceutical compositions comprising any of the products of the invention described herein, specifically any protein, nucleic acid or host cell, are also provided. In a preferred embodiment, the pharmaceutical composition is for immunotherapy.

Preferably, the TCR material of the invention is administered by injection, eg, intravenously. If the TCR material of the invention is a host cell expressing the TCR of the invention (or a functional variant thereof), the pharmaceutically acceptable carrier for cells for injection is any isotonic carrier, For example, normal saline (about 0.90% w / v NaCl aqueous solution, about 300 mOsm / L NaCl aqueous solution, or about 9.0 g NaCl / liter water), NORMOSOL R electrolyte (Abbott, Chicago, IL), PLASMA-LYTE A (Baxter, Deerfield, IL), about 5% dextrose in water, Ringer's emulsion may be included. In one embodiment, the pharmaceutically acceptable carrier is supplemented with human serum albumin.

For the purposes of the invention, the amount or dose of the TCR material of the invention administered (eg, if the TCR material of the invention is one or more cells, the number of cells) will be effective within a reasonable time. For example, the therapeutic effect on the subject or animal, or the preventive response effect should be sufficient. For example, the dose of the TCR material of the present invention is sufficient to bind to a cancer antigen for a period of about 2 hours or more, for example 12 to 24 hours or more, of administration, or detection, treatment or prevention of cancer. Must be enough to do. In certain embodiments, the period can be even longer. The dose is determined by the effectiveness of the particular TCR material of the invention and the condition of the animal (eg, human), and the weight of the animal (eg, human) being treated.

The pharmaceutical compositions of the invention, TCRs (including functional variants thereof), polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, or cell populations can be cancerous, HPV-infected, or HPV-positive premalignant. It is believed that it can be used in therapeutic or prophylactic methods. Without being bound by any particular theory, the TCRs (and functional variants thereof) of the invention are expressed by cells capable of mediating an immune response against target cells expressing the HPV antigen of the invention. If so, it is believed that the TCR (or related polypeptide or protein of the invention and functional variants thereof) specifically binds to the HPV antigen. In this regard, the invention is a method of treating or preventing a mammalian condition, wherein any of the pharmaceutical compositions, TCRs (and functional variants thereof), polypeptides, or proteins described herein. , Any nucleic acid or recombinant expression vector comprising a nucleic acid sequence encoding any of the TCRs (and functional variants thereof), polypeptides, proteins described herein, or the TCRs described herein. Any host cell or cell population containing (and its functional variants), a polypeptide, or a recombinant vector encoding a protein, in a mammalian cancer, HPV infection, or HPV-positive premalignant state. Provided are methods comprising administering to a mammal in an amount effective to treat or prevent.

Examples of pharmaceutically acceptable carriers or diluents useful in the present invention include stabilizers such as SPGA, carbohydrates (eg, sorbitol, mannitol, starch, sucrose, glucose, dextran), such as albumin or casein. Contains proteins, protein-containing agents such as bovine serum or sucrose, and buffers (eg, phosphate buffers).

As used herein, the terms "treatment" and "prevention" and the terms derived from them do not necessarily mean 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention that one of ordinary skill in the art will recognize as having potential benefits or therapeutic effects. In this regard, the methods of the invention can provide treatment or prevention of any level of any amount of mammalian condition. Moreover, the treatment or prevention provided by the methods of the invention may include the treatment or prevention of one or more conditions or symptoms of the condition being treated or prevented (eg, cancer). For example, treatment or prevention can include promoting tumor regression. Also, for the purposes of this specification, "prevention" may include delaying the onset of the condition, or delaying its symptoms or condition.

Also provided are methods of detecting the presence of a condition in a mammal. The methods are as follows: (i) TCRs (and functional variants thereof) of the invention described herein, polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, cell populations, antibodies or antigen binding thereof. By contacting a sample containing one or more cells of mammalian origin with either a moiety or a pharmaceutical composition to form a complex and to detect the complex of the complex. Detection is an indicator of the presence of a condition in a mammal, the condition including cancer, HPV16 infection, or HPV-positive pre-malignant condition.

For the method of the invention to detect a condition in a mammal, the cell sample is a whole cell, its lysate, or a portion of the whole cell lysate, eg, a nuclear or cytoplasmic fraction, a total protein fraction, or a nucleic acid fraction. Can be a sample containing.

For the purposes of the detection methods of the invention, contact can occur in vitro or in vivo with respect to mammals.

In addition, the complex can be detected by any number of methods known in the art. For example, the TCRs (and functional variants thereof), polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, cell populations, or antibodies or antigen-binding portions thereof of the invention described herein are detected. With possible labels, such as radioactive isotopes, fluorophores (eg, fluorescein isothiocyanate (FITC), phycoerythrin (PE)), enzymes (eg, alkaline phosphatase, horseradish peroxidase), and elementary particles (eg, gold particles). Can be labeled.

For the purposes of the methods of the invention, when a host cell or population of cells is administered, the cells can be allogeneic or autologous to the mammal.

With respect to the pharmaceutical use of the TCR material of the present invention described above, the cancers to be treated and / or prevented are acute lymphocytic cancer, acute myeloid leukemia, follicular rhombic myoma, bone cancer, brain cancer and breast cancer. , Anal cancer, anal duct or anal rectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, cervical cancer, bile sac, or thoracic cancer, nasal cancer, nasal cavity or middle ear cancer, oral Cancer, vaginal cancer, genital cancer, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer cancer, cervical cancer, gastrointestinal carcinoid tumor, glioma, hodgkin lymphoma, hypopharyngeal cancer, kidney Cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesotheloma, melanoma, multiple myeloma, nasopharyngeal cancer, non-hodgkin lymphoma, mesopharyngeal cancer, ovarian cancer, penis cancer, pancreatic cancer, peritoneum, reticulum and mesentery Cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small bowel cancer, soft tissue cancer, stomach cancer, testis cancer, thyroid cancer, uterine cancer, urinary tract cancer, and bladder cancer Any cancer, including cancer. Preferred cancers are cervical, oropharyngeal, anal, anal canal, anal rectum, vagina, vulva or penile cancer. Particularly preferred cancers are HPV-positive cancers, such as HPV16 or HPV18-positive cancers. Cancers most commonly associated with HPV 16/18 infection include cancers of the cervix, oropharyngeal, anal, anal canal, anal rectum, vagina, vulva and penis, but the methods of the invention are other anatomical. It may be used to treat any HPV-positive cancer, including cancers that occur in the area.

In general, the invention provides methods for treating a subject suffering from a tumor or tumor disease, comprising administration of the antigen recognition constructs, nucleic acids, vectors and / or host cells disclosed by the invention. Preferably, the subject is a subject in need of such treatment. In a preferred embodiment, the mammalian subject is preferably a human patient and suffers from an HPV-positive tumor or tumor disease.

The invention will be further described in the following examples with reference to the accompanying drawings and sequences, but is not limited thereto. For the purposes of the present invention, all references cited herein are incorporated by reference in their entirety.

FIG. 1 shows an example of a specific CD8 + T cell response to HLA-A * 02.01 restricted HPV16 E6 epitope TIHDIILECV in ABabDII mice. ABabDII mice were intraperitoneally immunized with (ip) E6 / 7 expressed adenovirus followed by subcutaneous (sc) 50 μg of E6 peptide emulsified in IFA with 50 μg of CpG ODN1826 or peptide / CpG / IFA mixture. Was administered alone. The presence of HPV-specific CD8 + T cells in the peripheral blood of immune animals was assessed by intracellular IFNγ staining 7 days after each boost. Stimulation with holball millistate acetate (PMA) and ionomycin was used as a positive control (+ ctrl). FIG. 2 shows an example of a specific CD8 + T cell response to the HLA-A201 restricted HPV16 E7 epitope TLGIVCPI in ABabDII mice. ABabDII mice were immunized subcutaneously (s.c.) at the base of the tail with 50 μg of CpG ODN1826 emulsified in IFA with 50 μg of E7 peptide. The presence of HPV-specific CD8 + T cells in the peripheral blood of immune animals was assessed by intracellular IFNγ staining 7 days after each boost. Stimulation with holball millistate acetate (PMA) and ionomycin was used as a positive control (+ ctrl). FIG. 3 shows a FACSort strategy (IFNγ capture assay) for specific CD8 + T cells against HLA-A * 02.01 restricted HPV16 E5, E6 and E7 epitopes. CD4 + T cells were depleted in spleen cells from response mice and restimulated with ^10-8 moles of each peptide for 10 days. For 5'RACE PCR, HPV-specific CD8 + T cells were sorted directly into RNA purification buffer using the IFNγ capture assay. FIG. 4 shows the HPV16 E6 and E7 TCR transduction of freshly prepared human PBL. 1 × 10 ⁶ freshly isolated or frozen hPBMCs were stimulated with anti-CD3 and anti-CD28 coated plates in the presence of 200 U / ml recombinant human interleukin 2. Addition of retrovirus containing supernatant and protamine sulfate, followed by spinocuration (first transduction) or preloading and spinoculation of virus onto a plate coated with retronectin (Takara) (second transduction). Transduction was performed 48 hours and 72 hours later. Transduction efficiency was assessed by staining with anti-CD8 and anti-mouse constant TCRβmAb. FIG. 5 shows the release of HPV16 E6 / E7 peptide-specific IFN-γ in human PBMC transduced with TCR; 5859TCR corresponds to SEQ ID NOs: 34 and 35; 5843B14TCR corresponds to SEQ ID NOs: 36 and 37; The 14800 TCR corresponds to SEQ ID NOs: 43 and 44. IFNγ production was measured by enzyme-bound immunoadsorption assay after 16-hour co-culture of 1 × 10 ⁴ TCR transduced T cells and 1 × 10 ⁴ peptide-loaded T2 cells. Stimulation with holball millistate acetate (PMA) and ionomycin was used as a positive control (max). ut: Non-transduced PBMC. FIG. 6 shows the HPV16 E6 IFN-γ release of TCR transduced human PBMCs in titrated E6-specific peptides and CASKI cell lines. The left panel shows the TCR of SEQ ID NO: 14/15 (T3) and the right panel shows the TCR of SEQ ID NO: 16/17 (T4). IFNγ production was measured by enzyme-bound immunoadsorption assay after 16-hour co-culture of 1x10 ⁴ TCR transduced T cells with 1x10 ^{4 peptide-loaded T2 cells or 1x10 4 CASKI} cell lines. Stimulation with holball millistate acetate (PMA) and ionomycin was used as a positive control (P + I). HLA-transduced cervical cancer cell line SiHa cell line and head and neck cancer cell line SCC-090 and SCC-152 also showed IFNγ release comparable to the recognition of CASKI cells (data not shown). FIG. 7 shows the HPV16 E5 transduction of freshly prepared human PBL and the HPV16 E5 peptide-specific IFN-γ release of human PBMC transduced with TCR. As the TCR, the TCR of SEQ ID NO: 12/13 (T2) was used. 1 × 10 ⁶ freshly isolated or frozen hPBMCs were stimulated with anti-CD3 and anti-CD28 coated plates in the presence of 200 U / ml recombinant human interleukin 2. By addition of retrovirus containing supernatant and protamine sulfate, followed by spinoculation (first transduction) or preloading and spinoculation of virus on retronectin (Takara) coated plates (second transduction). Transduction was performed after 48 and 72 hours. Transduction efficiency was assessed by staining with anti-CD8 and anti-mouse constant TCRβmAb. IFNγ production was measured by enzyme-bound immunoadsorption assay after 16 hours of co-culture of 1x10 ⁴ TCR transduced T cells and 1x10 ⁴ peptide-loaded T2 cells. Stimulation with holball millistate acetate (PMA) and ionomycin was used as a positive control (P + I).

SEQ ID NOs: 1-8: HPV16 and 18 epitopes to which the TCRs of the invention bind.
SEQ ID NOs: 9-28: The α-chain and β-chain CDR3 sequences of the TCR of the present invention are shown.
SEQ ID NOs: 29-48: The full-length variable sequences of the α-chain and β-chain of the TCR of the present invention are shown.

HPV epitopes used for immunization of animals:

ABabDIIマウスにおけるCTL反応性を有するペプチドエピトープ

Peptide epitope with CTL reactivity in ABabDII mice

(Example 1) T cell receptor T1 that recognizes HPV16-E5 epitope YIIFVYIPL

CDR１配列を太字で示す
CDR２配列を二重下線で示す
CDR３配列を一重下線で示す

CDR1 array shown in bold
CDR2 array is double underlined
CDR3 array is single underlined

Full length variable chain sequence (CDR3 sequence is underlined)

TRAV13-1 * 01-CAASSYNQGGKLIF-TRAJ23 * 01 (SEQ ID NO: 29)

TRAV26-2 * 01-CILRDVNAGGTSYGKLTF-TRAJ52 * 01 (SEQ ID NO: 30)

TRBV12-3 * 01-CASSLLSSYNEQFF-TRBD2 * 01-TRBJ2-3 * 01 (SEQ ID NO: 31)

(Example 2) T cell receptor T2 that recognizes HPV 16-E5 epitope YIIFVYIPL

Full length variable chain sequence (CDR3 sequence is underlined)

TRAV12-2 * 02-CAVNVDFNKFYF-TRAJ21 * 01 (SEQ ID NO: 32)

TRBV4-1 * 01-CASSQDWNNEQFF-TRBD1 * 01-TRBJ2-1 * 01 SEQ ID NO: 33

(Example 3) HPV 16-E6 epitope TI HDIILE CV-recognizing T cell receptor T3

Full length variable chain sequence (CDR3 sequence is underlined)

TRAV20 * 02-CAVQANRGSTLGRLYF-TRAJ18 * 01 SEQ ID NO: 34

TRBV28 * 01-CASSLWGRLAKNIQYF-TRBD1 * 01-TRBJ2-4 * 01 SEQ ID NO: 35

(Example 4) T cell receptor T4 that recognizes HPV 16-E6 epitope TIHDIILECV

Full length variable chain sequence (CDR3 sequence is underlined)

TRAV21 * 02-CAVRETSGSRLTF-TRAJ58 * 01 SEQ ID NO: 36

TRBV28 * 01-CASSFWGRSTDTQYF-TRBD1 * 01-TRBJ2-3 * 01 SEQ ID NO: 37

(Example 5) HPV 16-E6 epitope TI HDIILE CV-recognizing T cell receptor T5

Full length variable chain sequence (CDR3 sequence is underlined)

TRAV17 * 01-CATVSTDSWGKKLQF-TRAJ24 * 02 SEQ ID NO: 38

TRBV10-3 * 02-CAISDSNGINIQYF-TRBD2 * 02-TRBJ2-4 * 01 SEQ ID NO: 39

TRBV28 * 01-CASSLWGRAGKDTQYF-TRBD2 * 02-TRBJ2-3 * 01 SEQ ID NO: 40

(Example 6) HPV 16-E6 epitope T cell receptor T6 that recognizes KLPQLCTEL

Full length variable chain sequence (CDR3 sequence is underlined)

TRAV8-6 * 01-CAVSLNSGNTPLVF-TRAJ29 * 01 SEQ ID NO: 41

TRBV20-1 * 01-CSARDLAGNTGELFF-TRBD2 * 01-TRBJ2-2 * 01 SEQ ID NO: 42

(Example 7) T cell receptor T7 that recognizes the HPV 16-E7 epitope TLGIV PPI

Full length variable chain sequence (CDR3 sequence is underlined)

TRAV30 * 01-CGTGTDSWGKLQF-TRAJ24 * 02 SEQ ID NO: 43

TRBV12-4 * 01-CASSPGLAGGEQFF-TRBD2 * 02-TRBJ2-1 * 01 SEQ ID NO: 44

(Example 8) T cell receptor T8 that recognizes the HPV 16-E7 epitope TLGIVCPI

Full length variable chain sequence (CDR3 sequence is underlined)

TRAV22 * 01-CAVEPNSGNTPLVF-TRAJ29 * 01 SEQ ID NO: 45

TRBV7-2 * 02 or 03-CASSLIISYNEQFF-TRBJ2-1 * 01 SEQ ID NO: 46

(Example 9) T cell receptor T9 that recognizes the HPV 16-E7 epitope TLGIV PPI

Full length variable chain sequence (CDR3 sequence is underlined)

TRAV38-2 / DV-8 * 01-CAYRSAPYSGAGSYQLTF-TRAJ28 * 01 SEQ ID NO: 47

TRBV4-2 * 01-CASSQAPGLAGAEQYF-TRBD2 * 02-TRBJ2-7 * 01 SEQ ID NO: 48

(Example 10) Interferon gamma release by TCR T2, T3 and T4 transduced cell lines

6 and 7 show T cell lines transduced with TCR T3 and T4 (FIG. 6) and T2 (FIG. 7). Results are shown in the drawings and description of the drawings.

peptide: peptide
FACsorted population for TCR-specific 5'RACE PCR: FACsorted population for TCR-specific 5'RACE PCR
Donor: Donor

Claims (14)

An antigen recognition construct that specifically binds to a human papillomavirus (HPV) antigen, said antigen recognition construct .
I.a. Complementarity determining regions (CDR) 1 (DSASNY), CDR2 (IRSNVGE) and CDR3 (CAASSYNQGGKLIF) sequences contained in SEQ ID NO: 29, and
b. CDR1 (SGHNS), CDR2 (FNNNVP) and CDR3 (CASSLLSSYNEQFF) sequences contained in SEQ ID NO: 31, or
II. a. CDR1 (TISGTDY), CDR2 (GLTSN) and CDR3 (CILRDVNAGGTSYG KLTF) sequences contained in SEQ ID NO: 30 and
b. CDR1 (SGHNS), CDR2 (FNNNVP) and CDR3 (CASSLLSSYNEQFF) sequences contained in SEQ ID NO: 31, or
III. a. CDR1 (DRGSQS), CDR2 (IYSNGD) and CDR3 (CAVNVDFNKFYF) sequences contained in SEQ ID NO: 32, and
b. CDR1 (MGHRA), CDR2 (YSYEKL) and CDR3 (CASSQDWNNEQFF) sequences contained in SEQ ID NO: 33, or
IV. a. CDR1 (VSGLRG), CDR2 (LYSAGEE) and CDR3 (CAVQANRGSTLGRLYF) sequences contained in SEQ ID NO: 34, and
b. CDR1 (MDHEN), CDR2 (SYDVKM) and CDR3 (CASSLWGRLAKNIQYF) sequences contained in SEQ ID NO: 35, or
V. a. CDR1 (DSAIYN), CDR2 (IQSSQRE) and CDR3 (CAVRETSGSRLTF) sequences contained in SEQ ID NO: 36, and
b. CDR1 (MDHEN), CDR2 (SYDVKM) and CDR3 (CASSFWGRSTDTQYF) sequences contained in SEQ ID NO: 37, or
VI. a. CDR1 (TSINN), CDR2 (IRSNERE) and CDR3 (CATVSTDSWGKLQF) sequences contained in SEQ ID NO: 38, and
b. CDR1 (ENHRY), CDR2 (SYGVKD) and CDR3 (CAISDSNGINIQYF) sequences contained in SEQ ID NO: 39, or
VII. a. CDR1 (TSINN), CDR2 (IRSNERE) and CDR3 (CATVSTDSWGKLQF) sequences contained in SEQ ID NO: 38, and
b. CDR1 (MDHEN), CDR2 (SYDVKM) and CDR3 (CASSFWGRSTDTQYF) sequences contained in SEQ ID NO: 40, or
VIII. a. CDR1 (SSVSVY), CDR2 (YLSGSTLV) and CDR3 (CAVSLNSGNTPLVF) sequences contained in SEQ ID NO: 41, and
b. CDR1 (DFQATT), CDR2 (SNEGSKA) and CDR3 (CSARDLAGNTGELFF) sequences contained in SEQ ID NO: 42, or
IX. a. CDR1 (KALYS), CDR2 (LLKGGEQ) and CDR3 (CGTGTDSWGKLQF) sequences contained in SEQ ID NO: 43, and
b. CDR1 (SGHDY), CDR2 (FNNNVP) and CDR3 (CASSPGLAGGEQFF) sequences contained in SEQ ID NO: 44, or
X.a. CDR1 (DSVNN), CDR2 (IPSGT) and CDR3 (CAVEPNSGNTPLVF) sequences contained in SEQ ID NO: 45, and
b. CDR1 (SGHTA), CDR2 (FQGNS) and CDR3 (CASSLIISYNEQFF) sequences contained in SEQ ID NO: 46, or
XI. a. CDR1 (TSESDYY), CDR2 (QEAYKQQ) and CDR3 (CAYRSAPYSGAGSYQLTF) sequences contained in SEQ ID NO: 47, and
b. CDR1 (LGHNA), CDR2 (YNFKEQ) and CDR3 (CASSQAPGLAGAEQYF) sequences contained in SEQ ID NO: 48,
Antigen recognition constructs, including. The antigen recognition construct according to claim 1, wherein the antigen recognition construct is an antibody or a fragment thereof, or a T cell receptor (TCR) or a fragment thereof. The antigen recognition construct is a TCR or a fragment of the TCR composed of a TCRα chain and a TCRβ chain, wherein the TCR is SEQ ID NO: 29 and 31, or SEQ ID NOs: 30 and 31, or SEQ ID NOs: 32 and 33, or SEQ ID NO: 34. And 35, or SEQ ID NOs: 36 and 37, or SEQ ID NOs: 38 and 39, or SEQ ID NOs: 38 and 40, or SEQ ID NOs: 41 and 42, or SEQ ID NOs: 43 and 44, or SEQ ID NOs: 45 and 46, or SEQ ID NOs: 47. The antigen recognition construct according to claim 1 or 2, comprising the variable sequence shown in 48. A nucleic acid encoding the antigen recognition construct according to any one of claims 1 to 3. A vector containing the nucleic acid according to claim 4. A host cell comprising the antigen recognition construct according to any one of claims 1 to 3, the nucleic acid according to claim 4, or the vector according to claim 5. The host cell according to claim 6, wherein the host cell is a CD4 or CD8 positive T cell, T lymphocyte, or lymphocyte. The antigen recognition construct according to any one of claims 1 to 3, the nucleic acid according to claim 4, the vector according to claim 5, or the vector according to claim 6 or 7. Host cell. The antigen recognition construct, nucleic acid, or vector, or host cell of claim 8, for use in the diagnosis, prevention, and / or treatment of proliferative or infectious diseases, including malignant or benign tumor diseases. .. The antigen recognition construct, or nucleic acid, or vector, or host cell according to claim 8 or 9, wherein the pharmaceutical use is an immunotherapy comprising adoptive T cell therapy. An in vitro method for the production of cell lines expressing HPV-specific antigen recognition constructs (ARC).
a. The process of providing suitable host cells,
b. The process of providing a genetic construct that encodes ARC,
The ARC
I.a. Complementarity determining regions (CDR) 1 (DSASNY), CDR2 (IRSNVGE) and CDR3 (CAASSYNQGGKLIF) sequences contained in SEQ ID NO: 29, and
b. CDR1 (SGHNS), CDR2 (FNNNVP) and CDR3 (CASSLLSSYNEQFF) sequences contained in SEQ ID NO: 31, or
II. a. CDR1 (TISGTDY), CDR2 (GLTSN) and CDR3 (CILRDVNAGGTSYG KLTF) sequences contained in SEQ ID NO: 30 and
b. CDR1 (SGHNS), CDR2 (FNNNVP) and CDR3 (CASSLLSSYNEQFF) sequences contained in SEQ ID NO: 31, or
III. a. CDR1 (DRGSQS), CDR2 (IYSNGD) and CDR3 (CAVNVDFNKFYF) sequences contained in SEQ ID NO: 32, and
b. CDR1 (MGHRA), CDR2 (YSYEKL) and CDR3 (CASSQDWNNEQFF) sequences contained in SEQ ID NO: 33, or
IV. a. CDR1 (VSGLRG), CDR2 (LYSAGEE) and CDR3 (CAVQANRGSTLGRLYF) sequences contained in SEQ ID NO: 34, and
b. CDR1 (MDHEN), CDR2 (SYDVKM) and CDR3 (CASSLWGRLAKNIQYF) sequences contained in SEQ ID NO: 35, or
V. a. CDR1 (DSAIYN), CDR2 (IQSSQRE) and CDR3 (CAVRETSGSRLTF) sequences contained in SEQ ID NO: 36, and
b. CDR1 (MDHEN), CDR2 (SYDVKM) and CDR3 (CASSFWGRSTDTQYF) sequences contained in SEQ ID NO: 37, or
VI. a. CDR1 (TSINN), CDR2 (IRSNERE) and CDR3 (CATVSTDSWGKLQF) sequences contained in SEQ ID NO: 38, and
b. CDR1 (ENHRY), CDR2 (SYGVKD) and CDR3 (CAISDSNGINIQYF) sequences contained in SEQ ID NO: 39, or
VII. a. CDR1 (TSINN), CDR2 (IRSNERE) and CDR3 (CATVSTDSWGKLQF) sequences contained in SEQ ID NO: 38, and
b. CDR1 (MDHEN), CDR2 (SYDVKM) and CDR3 (CASSFWGRSTDTQYF) sequences contained in SEQ ID NO: 40, or
VIII. a. CDR1 (SSVSVY), CDR2 (YLSGSTLV) and CDR3 (CAVSLNSGNTPLVF) sequences contained in SEQ ID NO: 41, and
b. CDR1 (DFQATT), CDR2 (SNEGSKA) and CDR3 (CSARDLAGNTGELFF) sequences contained in SEQ ID NO: 42, or
IX. a. CDR1 (KALYS), CDR2 (LLKGGEQ) and CDR3 (CGTGTDSWGKLQF) sequences contained in SEQ ID NO: 43, and
b. CDR1 (SGHDY), CDR2 (FNNNVP) and CDR3 (CASSPGLAGGEQFF) sequences contained in SEQ ID NO: 44, or
X.a. CDR1 (DSVNN), CDR2 (IPSGT) and CDR3 (CAVEPNSGNTPLVF) sequences contained in SEQ ID NO: 45, and
b. CDR1 (SGHTA), CDR2 (FQGNS) and CDR3 (CASSLIISYNEQFF) sequences contained in SEQ ID NO: 46, or
XI. a. CDR1 (TSESDYY), CDR2 (QEAYKQQ) and CDR3 (CAYRSAPYSGAGSYQLTF) sequences contained in SEQ ID NO: 47, and
b. CDR1 (LGHNA), CDR2 (YNFKEQ) and CDR3 (CASSQAPGLAGAEQYF) sequences contained in SEQ ID NO: 48,
Is included,
c. The step of introducing the gene construct into the appropriate host cell,
d. The step of expressing the gene construct in the appropriate host cell,
Including, how. The method according to claim 11, wherein the ARC is the antigen recognition construct according to any one of claims 1 to 3. The method according to claim 11 or 12, wherein the host cell is a mammalian T cell including a human. The method of any one of claims 11-13, further comprising purification of ARC from cells and optionally reconstruction of ARC fragments translated with T cells.

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